Characterization of a recently recognized developmental pathway of bile acid synthesis, which begins with 26-hydroxylation of cholesterol in many tissues ("extrinsic" pathway of bile acid synthesis) and generates monohydroxy bile acids that when present in excess induce a cholestatic syndrome, will be continued in liver cell culture and in animal models. Although the biologic role (s) of cholest-5-ene-3Beta,26-diol (26-hydroxycholesterol), a potent inhibitor of cholesterol and DNA synthesis, has not been fully defined, the wide organ distribution of the mRNA for the 26-hydroxylase and the presence of 26- hydroxycholesterol and/or its metabolites in amniotic fluid, meconium, and adult plasma and bile indicate that the metabolic pathway is continuously active. The proportion of monohydroxy bile acids is dependent on both the production rate of 26-hydroxycholesterol and the activity of the 7alpha-hydroxylase that is essential for its metabolism to dihydroxy (chenodeoxycholic) and trihydroxy (cholic) bile acids. We propose that this enzyme differs from the cholesterol 7alpha-hydroxylase found in adults and that its activity normally.increases in early life. Delay in maturation explains the pathogenesis of some neonatal cholestatic syndromes. Sensitive and specific enzyme assays utilizing isotope ratio mass spectrometry will permit the characterization of this step and of its relationship to 7alpha-hydroxylation of cholesterol and other key enzymatic steps in bile acid synthesis in Hep G2 cells and in fetal, neonatal, weaned, and adult rabbits and hamsters. The regulation of 26-hydroxylase activity will be studied in rabbit liver using a labeled sterol 26-hydroxylase cDNA probe and the role of progesterone and other steroid hormones in regulating bile acid production will be studied in cell culture. Kinetic studies of pool size and turnover of 26-hydroxycholesterol will permit quantitative estimates of its contribution to bile acid production. Validation of the enzyme assays will permit their use in defining the pathogenesis of familial cholestatic syndromes and those occurring with hyperalimentation and drug therapy.